GENE SILENCING IN SKIN USING SELF-DELIVERY siRNA DELIVERED BY A MESO DEVICE

ABSTRACT

The present invention is drawn to a low-cost “meso” device that is able to effectively deliver functional self-delivery siRNA to subject and inhibit expression of a gene in the subject as well as related methods. In particular, a method of transdermally delivering nucleic acid material to a subject is provided. The method includes adapting a motorized meso machine for delivery of nucleic acid material; introducing nucleic acid material into a chamber of the motorized meso machine; and contacting the motorized meso machine to a skin surface of a subject for a period of time sufficient to deliver the nucleic acid material into the skin surface of the subject.

This application is a continuation of U.S. patent application Ser. No.15/668,606, filed Aug. 3, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/181,582, filed Feb. 14, 2014, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 61/764,928,filed Feb. 14, 2013, and U.S. Provisional Patent Application Ser. No.61/887,519, filed Oct. 7, 2013, each of which are incorporated herein byreference.

BACKGROUND

Of the 7,000 known monogenic disorders, approximately 2,000 affect theskin. While most of these are individually rare, together they representa significant healthcare burden and afflict up to 1% of the population.For most of these disorders, there are no effective treatments thattarget the root cause of the problem. Nucleic acid therapies, includingsiRNAs, are a potential way to modify expression of disease genes in acontrolled fashion, and hold real promise for improving patient lives.While traditional “small molecule” approaches to drug development havebeen a successful model for large pharmaceutical companies, the cost (onthe order of a $1 billion) and the length of development time (10-12years) limit their usefulness is rare inherited skin disorder.Identification of potent and selective siRNAs with limited off-targeteffects is now routine in many laboratories and the cost and timeinvolved is a fraction of what is required for small molecule drugdevelopment. The missing piece in translating siRNA technology to theclinic is a robust, reproducible, economical and “patient-friendly”(i.e., little or no pain) delivery platform. Substantial effort has beeninvested in a variety of delivery technologies, with increasing success.However, the complexity and cost may limit clinical translation andpatient compliance. For example, the first administration of siRNA toskin, and the first siRNA to target a mutant gene, was for pachyonychiacongenita, a rare genodermatoses caused by mutant keratin alleles. Theintradermal injection of TD101 siRNA (targets a single nt mutation[N171K] in the keratin 6a gene) resulted in improvement in thekeratoderma and lesion pain, but the painfulness of the intradermalinjection necessitated use of oral pain medication and a regional nerveblock and prevented further enrollment in the clinical trial.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows a handheld motorized meso/microneedles array device.

FIG. 1B shows the inner reservoir of the motorized microneedle arraycartridge array contains 300 μL of a red dye solution for visualization,and shows the needles are set to protrude 0.1 mm beyond the edge of thechamber.

FIG. 1C shows channels intermittently located between the needles allowflow of solution onto the surface of the skin during treatment.

FIG. 2A shows Cy3 labeled sd siRNA distribution in mouse flank skintreated by intradermal injection with a motorized microneedle arraydevice loaded with 50 μL 0.1 mg/mL Cy3-labeled sd-siRNA. These sectionsof hairless mouse flank skin show breaches in the epidermis center ofthe injection site. The individual images were stitched together usingICE software.

FIG. 2B shows magnification (10×) of the meso-treated andintradermally-injected skin.

FIG. 2C shows further magnification of images from 2B showing diffusionof the Cy3-labeled siRNA in the epidermis originating from the needlepenetration site (arrow). Left panels: 4′6-diamidino-2-phenylindole andCy3 overlay; right panels: Cy3 alone.

FIG. 2D shows distribution of fluorescently labeled sd-siRNA in humanabdominoplasty skin. Skin breaches due to penetration of the motorizedmicroneedles are seen in their entirety using a ×5 objective.Intradermally injected skin (50 μL of 0.1 mg/ml Cy3-labeled sd-snRNA)was similarly sectioned and imaged.

FIG. 2E shows magnification of treated skin.

FIG. 2F shows further magnification of the images from 2E showsdiffusion of the Cy3-labeled sd-siRNA in the epidermis originating fromthe needles penetration site (arrow), whereas low levels of fluorescenceare observed in the epidermis following intradermal injection. Leftpanels: 4′6-diamidino-2-phenylindole and Cy3 overlay; right panels: Cy3alone. Nuclei are visualized by 4′6-diamidino-2-phenylindole stain(blue). Scale bar=200 μm.

FIG. 3A shows a graphical representation of data showing thatmeso-assisted delivery of sd-siRNA inhibits targeted reporter geneexpression from the following procedure. Hairless tg CBL/hMGFP mouseflank skin was treated daily with the meso device loaded with 50 μL 10mg/mL CBL3 sd-siRNA or non-specific control sd-siRNA (sd-TD101) for 10days. On day 11, the mice were sacrificed and the treated skin wasexcised for RTqPCR analysis and fluorescence imaging. Total RNA wasisolated from the epidermis of the excised skin, reverse transcribed andhMGFP mRNA levels (relative to K14) were quantified in triplicate byqPCR. Bars indicate standard error.

FIG. 3B shows representative fluorescence images (bottom) with brightfield overlay (top) of frozen skin sections (10 μm) prepared fromtreated mice showed knockdown of hMGFP signal fluorescence signal in theskin treated with specific sd-siRNA over control sd-siRNA. Nuclei arevisualized by 4′,6-diamidino-2-phenylindole stain (blue). Scale bar is100 μm.

FIG. 4A-4C show confocal fluorescence imaging of Cy3-labeled sd-siRNA inhuman skin xenografts. A,B. Following meso assisted delivery (30-60 min)of 100 μL 0.5 mg/mL Cy3-labeled sd-siRNA (in Phosphate Buffered Saline,hereinafter “PBS”) into human skin, reflectance (panel A) and redfluorescence (panel B, C) were imaged with the Lucid Vivascope system)

FIG. 5A shows a graphical representation of a procedure where the mesochamber was loaded with the indicated volume of PBS solution and appliedto mouse flank skin at the 0.1 mm depth setting for 10 s. The solutionremaining in the chamber and on the surface of the skin was collectedand measured (red bars).

FIG. 5B shows images from the following procedure. To confirm the volumedelivered as described in A, 50 μL of labeled siRNA was loaded in thechamber and an equivalent volume of the predicted delivery volume usingthe meso device (16 μL) was intradermally injected adjacent to the mesotreatment site and imaged with the IVIS Lumina II.

FIG. 6A shows distribution of fluorescently-labeled siRNA in frozensections prepared from meso-treated skin, specifically, Cy3-labeledsiRNA distribution in human skin. Human abdominoplasty skin was treatedwith the meso device loaded with 50 μL 0.1 mg/mL Cy3-labeled sd-siRNA(Accell) or injected intradermally with 50 μL of the same solution. Skinbreaches due to meso needle penetration are seen in their entirety usinga 5× objective as well as a distribution gradient in signal from thedelivery site. Intradermally-injected skin was sectioned to the centerof the injection and similarly imaged. The individual images (stitchedtogether using ICE software, see Materials and Methods) show similarlevels of fluorescence when comparing the meso-treated and ID-injectedskin sites.

FIG. 6B shows magnification (10× objective) of the meso-treated skinshowed diffusion of the Cy3-labeled siRNA in the epidermis originatingfrom the needle penetration site, whereas low levels of fluorescence wasobserved in the epidermis following intradermal injection. Left panels:DAPI and Cy3 overlay; right panels: Cy3 alone. Scale bar=400 μm.

SUMMARY

As set forth herein, the present invention is drawn to a low-cost “meso”device that is able to effectively deliver functional self-deliverysiRNA to a subject and inhibit expression of a gene in the subject aswell as related methods. Accordingly, a method of transdermallydelivering nucleic acid material to a subject is provided. The methodincludes adapting a motorized meso machine for delivery of nucleic acidmaterial; introducing nucleic acid material into a chamber of themotorized meso machine; and contacting the motorized meso machine to askin surface of a subject for a period of time sufficient to deliver thenucleic acid material into the skin surface of the subject.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENT(S)

Before the present devices, formulations, systems and methods for thedelivery and use of nucleic acid materials are disclosed and described,it is to be understood that this invention is not limited to theparticular process steps and materials disclosed herein, but is extendedto equivalents thereof, as would be recognized by those ordinarilyskilled in the relevant arts. It should also be understood thatterminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting.

It should be noted that, the singular forms “a,” “an,” and, “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, reference to “a nucleic acid material” includesreference to one or more of such nucleic acids materials, and referenceto “the motorized meso machine” includes reference to one or more ofsuch motorized meso machines.

As used herein, “subject” refers to a mammal in having a condition forwhich rapamycin is a therapeutically effective treatment. In someaspects, such subject may be a human.

As used herein, the term “motorized meso machine” or “motorized mesodevice” are used interchangeably and refers to a motorized microneedledevice that when placed on a skin surface can cause the microneedles topenetrate a skin surface due to vibration caused by the device. Suchdevices are well known in the cosmetic and dermal arts. A commerciallyavailable example of such a device is the Tiple-M or TriM by BomtechElectronic Co, Seoul, South Korea).

As used herein, the term “about” is used to provide flexibility to anumerical range endpoint by providing that a given value may be “alittle above” or “a little below” the endpoint.

The terms, “comprises,” “comprising,” “containing” and “having” and thelike can have the meaning ascribed to them in U.S. Patent law and canmean “includes,” “including,” and the like, and are generallyinterpreted to be open ended terms. The terms “consisting of” or“consists of” are closed terms, and include only the components,structures, steps, or the like specifically listed in conjunction withsuch terms, as well as that which is in accordance with U.S. Patent law.“Consisting essentially of” or “consists essentially of” have themeaning generally ascribed to them by U.S. Patent law. In particular,such terms are generally closed terms, with the exception of allowinginclusion of additional items, materials, components, steps, orelements, that do not materially affect the basic and novelcharacteristics or function of the item(s) used in connection therewith.For example, trace elements present in a composition, but not affectingthe compositions nature or characteristics would be permissible ifpresent under the “consisting essentially of” language, even though notexpressly recited in a list of items following such terminology. Whenusing an open ended term, like “comprising” or “including,” it isunderstood that direct support should be afforded also to “consistingessentially of” language as well as “consisting of” language as ifstated explicitly.

As used herein, compounds, formulations, or other items may be presentedin a common list for convenience. However, these lists should beconstrued as though each member of the list is individually identifiedas a separate and unique member. Thus, no individual member of such listshould be construed as a de facto equivalent of any other member of thesame list solely based on their presentation in a common group withoutindications to the contrary.

Concentrations, amounts, and other numerical data may be expressed orpresented herein in a range format. It is to be understood that such arange format is used merely for convenience and brevity and thus shouldbe interpreted flexibly to include not only the numerical valuesexplicitly recited as the limits of the range, but also to include allthe individual numerical values or sub-ranges encompassed within thatrange as if each numerical value and sub-range is explicitly recited. Asan illustration, a numerical range of “about 0.5 to 10 g” should beinterpreted to include not only the explicitly recited values of about0.5 g to about 10.0 g, but also include individual values and sub-rangeswithin the indicated range. Thus, included in this numerical range areindividual values such as 2, 5, and 7, and sub-ranges such as from 2 to8, 4 to 6, etc. This same principle applies to ranges reciting only onenumerical value. Furthermore, such an interpretation should applyregardless of the breadth of the range or the characteristics beingdescribed.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, representativemethods, devices, and materials are described below.

Reference will now be made in detail to preferred embodiments of theinvention. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that it is not intended tolimit the invention to those preferred embodiments. To the contrary, itis intended to cover alternatives, variants, modifications, andequivalents as may be included within the spirit and scope of theinvention as defined by the appended claims.

Despite the development of potent siRNA molecules that effectivelytarget genes responsible for skin disorders, translation to the clinichas been hampered by the difficulty of efficient delivery through thestratum corneum barrier into the live skin cells. Although intradermalinjection of siRNA using hypodermic needles results in reproducible genesilencing, this approach is limited by the size of a single injectionand is painful. The use of microneedle arrays is a less painful methodfor siRNA delivery, but limited payload capacity limits this approach tohighly potent molecules. In the present case, a device that combinesuseful elements of both hypodermic needles and array technologies wasused to effectively deliver functional siRNA to mouse and human skin.This commercially-available device utilizes an array of vibrating,adjustable-height needles, facilitating delivery of cargo solutionthrough the stratum corneum with little to no pain. Treatment of bothhuman and murine skin resulted in distribution throughout the treatedskin (including the epidermis). Efficient silencing (58% reduction) ofreporter gene expression was achieved in a transgenic reporter mouseskin model.

siRNAs are promising agents for treating monogenic skin disordersparticularly those caused by dominant mutations, if delivery concernscan be overcome. The use of siRNAs as therapeutics has made substantialprogress in recent years and clinical trials are underway for treatmentof a variety of disorders in eye, liver, kidney and skin. Due to theaccessibility of skin, direct injection of “naked” nucleic acids hasbeen suggested as the most simple, safe and efficient delivery method.However, direct injections are limited to a highly localized region ofthe epidermis coincident to the injection site, and large number ofinjections may be needed to achieve uniform delivery and a therapeuticoutcome.

The first siRNA used in skin, TD101, targeted a mutant version (N171K)of keratin 6a, which is one of the mutations responsible for thedominant negative monogenic skin disorder pachyonychia congenita (PC).TD101 was also the first siRNA used in the clinic to target a mutantgene. Improvements in PC symptoms were observed at the plantar site ofsiRNA intradermal injection in the single patient initially enrolled inthe study, but not the paired injection site on the opposite foot thatreceived vehicle alone. Intradermal injections of either siRNA orvehicle alone were accompanied by severe pain, necessitating nerveblocks as well as oral pain medication on the treatment days. Additionalpatients were not enrolled due to the intense pain associated with theseinjections. The intense pain associated with intradermal injection leadsto the need to explore alternative “patient-friendly” deliverytechnologies (i.e. effective delivery of functional siRNA with little orno pain).

Multiple physical approaches have been reported in the literature tofacilitate delivery across the stratum corneum barrier includingultrasound, erbium:YAG laser, gene gun (ref), iontophores is,electroporation and microneedles. Once the siRNA passes the stratumcorneum barrier, however, the affected cells must still internalize thesiRNA in a manner that allows for incorporation into the RNA-inducedsilencing complex (RISC). Naked siRNA is not normally taken up bykeratinocytes in the absence of transfection unless the siRNAadministration is accompanied with pressure (“pressure-fection”). It hasbeen shown that covalent “self-delivery” modifications (includingDharmacon's Accell modifications), facilitate cellular uptake in vitroand in vivo without the need for tranfection reagents. Additionally, ithas been previously reported that administration of these self-deliverysiRNA by dissolvable microneedle arrays could reduce target geneexpression up to 50% in both mouse and human skin models. The nearly 60%reduction in target gene expression reported herein delivering sd-siRNAswith the meso device warrants additional study for use in patients andrepresents an alternative path to using microneedle arrays for deliveryacross the stratum corneum barrier.

With the above in mind, the present invention is drawn to a low-cost“meso” device that is able to effectively deliver functionalself-delivery siRNA to subject and inhibit expression of a gene in thesubject as well as related methods. Accordingly, a method oftransdermally delivering nucleic acid material to a subject is provided.The method includes adapting a motorized meso machine for delivery ofnucleic acid material; introducing nucleic acid material into a chamberof the motorized meso machine; and contacting the motorized meso machineto a skin surface of a subject for a period of time sufficient todeliver the nucleic acid material into the skin surface of the subject.The adapting of the motorized meso device can include adjusting thedepth of needle penetration for the device and/or adjusting theoscillation rate of the needles. It is noteworthy that it istheoretically possible that the “adapting” step of the claimed inventionmay not require any affirmative adjustment of the device, rather a merechecking of the settings of the device to assure that they are at thedesired setting for a given application. In one aspect, the needles ofthe motorized meso machine includes deliver the nucleic acid materialinto the skin surface at a depth of about 25 microns to about 3 mm.

In one embodiment, the nucleic acid material delivered by the motorizedmeso machine can be siRNA. In another embodiment, the nucleic acidmaterial can be sd-siRNA. In some aspects, it can be useful to utilizenucleic acid material that is suspended in a solution. In oneembodiment, the nucleic acid material can be suspected in a PBSsolution. Other solvents or liquid carriers known in the art to becompatible with nucleic acid material can also be used.

Once the motorized meso machine is applied loaded with the nucleic acidmaterial, the machine can contact the skin of a subject being treatedfor a period of time sufficient to allow for delivery of the nucleicacid. In some embodiments, the period of time of contacting can be for aperiod of about 5 seconds to about 20 seconds. In another embodiment,the period of time can be about 7 seconds to about 15 seconds. Thecontacting can be repeated in the same skin area or can be repeated onother skin areas, e.g. adjacent skin areas in order to provide thedesired delivery.

The disclosed invention provides a strong alternative to traditionalmicroneedles and hypodermic needle injections.

EXAMPLES

The following examples are provided to promote a more clearunderstanding of certain embodiments of the present invention, and arein no way meant as a limitation thereon. The compositions may besuitably modified by a person skilled in the art. The followingmaterials and methods were utilized in the Examples described herein.

Animals

Hairless mice (6-8 weeks old) were purchased from Charles RiverLaboratories (Wilmington, Mass.) and housed at TransDerm. Hairless TgCBL/hMGFP mice were generated by breeding Tg CBL/hMGFP mice on aHairless background and maintained at Stanford University. Animals weretreated according to the guidelines of the National Institutes of Health(NIH), TransDerm and Stanford University.

siRNA

Unmodifed specific (CBL3 and non-specific siRNAs (TD101 K6a3′UTR.1 NSC4and “self-delivery” Accell® specific (sd-CBL3 and non-specific (sd-TD101sd-CD44 and Cy3-labeled (ref)) siRNAs containing Dharmacon-proprietarymodifications allowing for cellular uptake without traditionaltransfection reagent) were synthesized by Thermo Fisher Scientific,Dharmacon Products (Lafayette, Colo.); Accell siRNAs are availablecommercially from this source.

Example 1—Delivery of siRNA Cargo into the Epidermis by the Meso Device

Despite the promise of siRNA for use as skin therapeutics, a majorobstacle is delivery of these molecules through the stratum corneumbarrier due to their size (˜13,000 MW) and polyanionic nature. Mesodevices can be used to deliver a variety of molecules across thisbarrier through direct penetration using vibrating needles that areprovided as a single use sterile disposable cartridge (FIG. 1). As thedepth of needle penetration can be adjusted, this device has thepotential to deliver cargo to different skin types ranging from thinmouse skin (<50 um) to thick human plantar skin (>1 mm). Furthermore,the ability to adjust depth allows for deposition in the epidermis,avoiding the pain nerve fibers that are prevalent in the dermis.

To determine the amount of potential cargo that can be delivered toskin, the meso device was loaded with 50, 100, 200 or 300 μL of PBSsolution. After application to murine flank skin for 10 s (set todeliver at a depth of 0.1 mm), the solution remaining in the meso deviceand on the surface of the skin was collected and measured and used tocalculate the total volume delivered. Once a threshold volume of 100 μLwas loaded into the device, adding additional solution did not result inincreased cargo delivery (FIG. 5A). Thus, in mouse flank skin, a maximaldelivered volume of 40 μL occurred when 100 μl of solution was loadedinto the cartridge. In order to confirm that delivery was occurring,murine flank skin was treated with the meso device loaded with 50 μL of0.1 mg/mL fluorescently-tagged sd-siRNA (Cy3-Accell siRNA, see Materialsand Methods). The calculated volume delivered by meso (16 μL) of thesame solution was then intradermally injected adjacent to themeso-treated area. Fluorescence was measured by in vivo imaging andresulted in similar intensities (FIG. 5B).

In order to visualize siRNA distribution in the skin, mice weresacrificed 1 h following treatment with the fluorescently-tagged siRNA,and the treated skin was embedded in OCT, sectioned (10 μm) and analyzedby fluorescence microscopy. Meso-assisted delivery resulted in agradient of fluorescently-tagged siRNA distribution throughout treatedarea of the skin with peak intensity observed at the site of needleinjection (FIG. 2A). Importantly, the bulk of fluorescent signal wasobserved migrating laterally through the epidermis from the needlepenetration site (FIG. 2B). Intradermal injection of thefluorescently-tagged siRNA also resulted in distribution of signalthroughout the dermis and epidermis (FIG. 2A and data not shown). Thedistribution of labeled sd-si-RNA in human skin was similarly analyzed(FIG. 2D). As in mouse skin, the fluorescent signal was observed in agradient pattern from the site of needle penetration including lateraldistribution through the epidermis (FIG. 2E, F). In contrast to thedistribution pattern observed upon intradermal injection in mouse flank(FIG. 2C), less labeled siRNA was detected in the epidermis of the humanskin following intradermal injection with a hypodermic needles (FIG. 2E,F), consistent with previous experiments in both human abdominal explantskin and mouse footpad skin.

Example 2—Silencing of CBL/hMGFP Reporter Gene in Transgenic MouseEpidermis

It has been previously reported silencing of a fluorescent reporter genein a tg mouse skin model (Tg CBL/hMGFP) after administration ofunmodified and self-delivery CBL3 siRNA by intradermal injection andmicroneedle application, respectively. In order to evaluate the abilityof the meso device to deliver functional sd-CBL3 siRNA in this model, TgCBL/hMGFP mouse flank skin was treated and reporter gene expressionanalyzed. Flank skin was treated every day for 10 days. On day 11, themice were sacrificed and flank skin was excised for RNA isolation andhistology. Reporter mRNA (CBL/hMGFP) levels were measured by RT-qPCR(FIG. 3A). A significant reduction (averaging 58±5%) of reporterexpression was detected in skin treated with the specific CBL3 sd-siRNAcompared to the contralateral flank skin treated with non-specificcontrol sd-siRNA (sd-CD44 siRNA). This experiment was repeated comparingCBL3 sd-siRNA to non-specific sd-TD101 siRNA with similar results (datanot shown). The decreased hMGFP levels were corraborated by fluorescencemicroscopy of CBL3 sd-siRNA-treated skin compared to control sd-siRNAtreatment (FIG. 3B). Fluorescence images of hMGFP were overlaid with4′,6-diamidino-2-phenylindole (DAPI) and bright field images to locatethe basal layer and stratum corneum.

Example 3—Distribution of Fluorescently-Tagged siRNA FollowingMeso-Assisted Delivery in Human Skin

In order to visualize delivery of sd-siRNA in a human model,freshly-obtained explant skin (from plastic surgery procedures) wastreated for 10 s with the meso device set at a depth of 100 μm. 50 μL of0.1 mg/mL Cy3-labeled sd-siRNA was delivered to skin for cryosectioningwhile 100 μL of 0.5 mg/mL Cy3-labeled sd-siRNA was delivered to skin forin vivo confocal imaging. For cryosectioning, the skin was embedded inOCT 1 h post-treatment, sectioned and imaged by fluorescent microscopy(FIG. 6A). Similar to delivery of labeled sd-siRNA to mouse skin, thefluorescent signal was observed in a gradient pattern from the site ofneedle penetration including diffusion laterally through the epidermis(FIG. 6B). Interestingly, lower signal was detected in the epidermis ofintradermally-injected skin (FIG. 6B and data not shown).

siRNA distribution was also visualized in human skin using confocalimaging. Skin removed during a rhytidectomy procedure was treated with100 μL 0.5 mg/mL Cy3-labeled sd-siRNA and imaged using the LucidVivascope 30-60 min post-treatment. Reflectance images collected at 658nm show the needle penetration site (FIG. 4A) at 26 μm depth. Redfluorescence (ex. 532 nm; em. 607 nm) at 59 μm depth shows radialdistrubution from the needle penetration side (FIG. 4B) and interactionwith individual cells (FIG. 4C).

Example 3—Meso-Assisted Delivery of sd-siRNA

A Motorized Meso Machine (Triple M) was adapted for delivery of siRNA tomouse and human skin. sd-siRNA solution (up to 300 μL) was introducedinto the chamber of the disposable meso needle cartridge using astandard P-200 pipet tip. For treating mice, a fold of skin was laidflat on a plastic surface and held in place with the tip of the mesodevice. With the meso device oriented vertically (perpendicular to thefold of skin) the device was turned on and held in place for 10 seconds.For treating human skin, fresh abdominal skin (obtained fromabdominoplasty procedure) was manually stretched and pinned to a corkplatform prior to treatment as described above.

Example 4—Histological Analysis of Fluorescently-Labeled sd-siRNADistribution in Murine and Human Skin

Cy3-Accell Non-Targeting siRNA (Dharmacon Products, Thermo FisherScientific, Lafayette, Colo.) was loaded into the chamber of the mesodevice (50 μL 0.1 mg/mL). Mouse flank skin or de-identified humanabdominal skin from an abdominoplasty procedure was treated as describedabove and imaged in an IVIS Lumina imaging system (Xenogen product fromCaliper LifeSciences, Alameda, Calif.) using the 535 nm excitation andDsRed emissions settings (1-10 s acquisition time). The data werequantified using Livinglmage software (Caliper LifeSciences) andpresented as an overlay with the brightfield data. Fluorescentbackground from an untreated area of the same animal or tissue samplewas subtracted and values were reported as radiant efficiency. Skin wasthen embedded in OCT and sectioned for analysis by fluorescencemicroscopy using a Zeiss Axio Observer Inverted Fluorescence Microscopeequipped with Cy3 and DAPI filter sets as previously described.

Example 5—Confocal Microscopy of Fluorescently-Labeled sd-siRNADistribution in Murine and Human Skin

Cy3-Accell Non-Targeting siRNA was loaded into the chamber of the mesodevice (100 μL 0.5 mg/mL). De-identified human facial skin from a facelift procedure was treated as described above and imaged using amodified Lucid VivaScope 2500 System (Lucid Inc., Rochester, N.Y.) 30-60min following treatment as previously described. Briefly, image z-stackswere generated by image acquisition at successive z-depths using nativeVivaScan software (v. VS008.01.09), and then post-processed using publicdomain Fiji java-based image processing software, Images were acquiredin reflectance mode using a 658 nm laser source, and duplicate stackswere acquired using a 532 nm excitation laser and with a long passfilter to collect 607 nm emission.

To increase the effective resolution of the VivaScope images, 10 nominalduplicate images were taken at each z-step, and these 10 image sets wereaveraged to produce z-step-averaged images, resulting in the final imagestack. Because in vivo imaging is influenced by respiration and otherminor subject motion, successive frames were co-registered using anaffine transform; distributed with Fiji software as the StackReg plugin)prior to any frame-averaging. Images were further intensity-scaled tomaximize contrast with the Fiji software using a global constraint suchthat the highest intensity 0.1% of image pixels in each frame werescaled to a pixel intensity value of 256.

Example 6—Meso-Assisted Delivery of sd-siRNAs and Analysis of GeneSilencing

Two cohorts of anesthetized tg-CBL/hMGFP mice were treated with 50 μL of10 mg/mL solution in PBS of either sd-CBL3 siRNA or a non-specificcontrol sd-siRNA (CD44 or TD101 sd-siRNA) as described above every dayfor 10 days. The day following the last treatment, mice were euthanizedand the treated area was excised for analysis by both fluorescencemicroscopy and RTqPCR as described in with the following modifications.The epidermis was separated from the dermis by incubation in dispase II(10 mg/mL in PBS, Roche, Indianapolis, Ind.) for 2-4 hours at 21° C.prior to RNA isolation.

Example 7—Histological Analysis of Treated Skin

To assess potential tissue damage due to penetration of the arraymicroneedles alone versus inflammation caused by deposition of sd-siRNA,PBS, or sd-siRNA (in PBS) was administered to hairless tg-CBL/hMGFP micewith MMNA device. The skin was harvested 24 hours after treatment andimmediately fixed in formalin and embedded in paraffin. Histology of thetreated skin revealed areas consistent with needle penetration andassociated skin damage. Acute inflammation was observed with prominentpolymorphonuclear infiltrate in the papillary dermis extending downinto, but not through the reticular dermal layer, primarily at eh siteof needle penetration through the epidermis but also throughout thedermis. Scattered macrophages and chronic inflammatory cells were alsopresent, consistent with classic wound healing, as would be expendedfrom a standard hypodermic needle injection. Inflammation was generallylocalized around wounded regions. There were no visual differences inwound response in skin treated with vehicle alone as compared with skintreated with TD101 or CBL3 sd-siRNA (data not shown), suggesting thatobserved acute inflammation is not due to the presence of sd-siRNA.

Example 8—Analysis and Discussion of the Use of Meso-Devices to Deliversd-siRNA's

Due to its accessibility, skin is an attractive target for siRNAtherapeutics, and direct injection of “naked” nucleic acids are thoughtas simple, safe, and efficient delivery method. However, directinjections are limited to a highly localized region of the epidermiscoincident with the injection site, and large number of injections maybe needed to achieve the uniform delivery required for a favorabletherapeutic outcome. Indeed, although some efficacy may result fromintradermal injection of siRNA, generally the efficacy can be limited tothe area immediately surrounding the plantar injection site. Furtherintradermal injections of either siRNA or vehicle alone are accompaniedby severe pain, necessitating nerve blocks as well as oral painmedication before treatment. This pain is likely due, at least in part,to the large volume (up to 2 ml) of drug injected into the lesion. Thehigh pressure required for siRNA delivery is also likely at leastpartially responsible for the intense pain experienced with theseinjections. Thus, the disclosed invention is an alternative“patient-friendly” (i.e. little or no pain) delivery technology.

For functional delivery, siRNA must not only transit the stratum corneumbarrier, but also be internalized into cells in a manner that allows forincorporation into the RNA-induced silencing complex (RISC). In additionto direct injection with hypodermic needle, multiple physical approacheshave been evaluated the reportedly facilitate delivery of nucleic acidsacross the stratum corneum barrier including ultrasound, erbium:YAGlaser, gene gun, iontophoresis, electroporation, microneedles, and nowmotorized microneedles. However, unmodified nucleic acids are notnormally taken up by keratinocytes in the absence of transfection agentsunless the administration is accompanied with pressure(“pressure-fection”). Covalent “sd” siRNA modifications (e.g.,Dharmacon's Accell modifications) facilitate a cellular uptake in vitroand in vivo without the need for transfection reagents. Administrationof sd-siRNA by dissolvable microneedle arrays can reduce target geneexpression up to 50% in both mouse and human skin models. The nearly 90%average reduction in target gene expression provided by the devices andtechniques disclosed herein exceeds with the threshold of 50% targetgene expression reported via the use of dissolvable microneedles.

The results set forth in the above examples indicate that disclosed mesodevices effectively deliver siRNA to relevant regions of the skin withan efficiency (up to 80% inhibition) that, if translatable to humansubjects, may offer relief to patients suffering from debilitatingmonogenic skin disorders. In contrast to this, direct injection ofunmodified siRNA with a hypodermic needle results in 33% decrease inreporter gene expression. Generally it is known that the use ofmicroneedles significantly decreases pain associated as compared withintradermal injections inhuman studies.

It has to be understood that the above-described various types ofcompositions, are only illustrative of preferred embodiments of thepresent invention. Numerous modifications and alternative arrangementsmay be devised by those skilled in the art without departing from thespirit and scope of the present invention and the appended claims areintended to cover such modifications and arrangements. Thus, while thepresent invention has been described above with particularity and detailin connection with what is presently deemed to be the most practical andpreferred embodiments of the invention, it will be apparent to those ofordinary skill in the art that variations including, may be made withoutdeparting from the principles and concepts set forth herein.

1. A method for delivering nucleic acid material for treatment of amonogenic skin disorder, the method comprising: introducing a nucleicacid material into a chamber of a motorized meso machine, the motorizedmeso machine comprising microneedles operatively coupled to the chamber;contacting the microneedles of the motorized meso machine to a skinsurface of a subject in need of the treatment; and actuating themotorized meso machine such that the microneedles are inserted into theskin surface of the subject for a period of time for delivering atherapeutically effective dose of the nucleic acid material from thechamber into the skin surface of the subject.
 2. The method of claim 1,wherein the nucleic acid material comprises siRNA or sd-siRNA configuredto silence a gene, a mutation in which results in the monogenicdisorder.
 3. The method of claim 1, wherein the monogenic disorder ispachyonychia congenita.
 4. The method of claim 5, wherein the nucleicacid material comprises TD101 siRNA.
 5. The method of claim 1, whereinthe steps of contacting the microneedles and actuating the motorizedmeso machine are repeated periodically on a same area on the skinsurface so as to periodically deliver the nucleic acid material to thesame area.
 6. The method of claim 1, wherein step of contacting themicroneedles and actuating the motorized meso machine are repeated on anadjacent skin area so as to deliver the nucleic acid material to theadjacent skin area.
 7. The method of claim 1, wherein the microneedlesare configured to deliver the nucleic acid material into the skinsurface at a depth in a range from 25 μm to 3 mm.
 8. The method of claim1, wherein the period of time is at about 5 seconds to about 20 seconds.9. The method of claim 1, wherein the subject is a human.
 10. The methodof claim 1, further comprising adjusting, following an insertion of themicroneedles into the skin surface, a rate of oscillation of themicroneedles by the motorized meso machine.
 11. A device for treatmentof a monogenic skin disorder, the device comprising: a motorized mesomachine having: a chamber configured to contain a volume of a thenucleic acid material; and microneedles operatively coupled to thechamber such that upon contacting the microneedles to a skin surface ofa subject and actuating the motorized meso machine for a period of time,the nucleic acid material flows from the chamber onto the skin surfaceand the microneedles are inserted into the skin surface to deliver apredetermined volume of the nucleic acid material from the chamber intothe skin surface of the subject at a predetermined depth.
 12. The deviceof claim 11, wherein the device is configured to oscillate themicroneedles at an adjustable a rate of oscillation.
 13. The device ofclaim 11, wherein microneedles have a length in a range from 25 μm to 3mm.
 14. The device of claim 11, wherein the predetermined depth to whichthe nucleic acid material is delivered is in a range from 25 μm to 3 mm.15. The device of claim 11, wherein the predetermined volume is in arange from 10 μl to 500 μl.
 16. The device of claim 11, wherein theperiod of time is in a range from 5 seconds to 20 seconds.
 17. Thedevice of claim 11, wherein the nucleic acid material comprises anucleic acid and an indicator.
 18. The device of claim 11, wherein thesubject is a mammal.
 19. The device of claim 11, wherein the nucleicacid material comprises siRNA or sd-siRNA.
 20. The device of claim 11,wherein the nucleic acid material is suspended in a solution comprisinga phosphate buffer.